We are studying control of globin gene expression in K562 human leukemia cells which, we have shown, synthesize embryonic and fetal hemoglobins, but not adult hemoglobin A, free Beta-chains, or Beta-globin RNA. The karyotype of this transformed cell line demonstrates triploidy with a translocation of varying length on the distal end of the short arm of one chromosome 11 (11p), near the locations proposed for the Epsilon-Gamma-Beta-globin gene complex, as well as the insulin gene and the c-Ha-ras-1 protooncogene. Using in situ hybridization of tritiumlabelled probes to K562 metaphase chromosomes, we localized these five genes to the proximal one-third of chromosome 11p. Since this localization differed from several reports, we performed extensive in situ hydridization studies with normal lymphocytes and showed that the Epsilon-Gamma-Beta-globin genes are located at somatic metaphase bands 11.2-12, while the ras and insulin genes are at band 14.1, identical to the results obtained with K562 cells. The results suggests these genes are distant from the K562 translocation breakpoint. To investigate whether DNA methylation was related to the lack of expression of the K562 Beta-globin gene we cultured cells in 5-azacytidine (5-AzaC), a cytidine analogue which causes hypomethylation of DNA. We detected, by S1 nuclease analysis, low but significant levels of Beta-globin mRNA following 5-AzaC treatment, while other globin mRNAs increased in abundance. The DNA of K562 cells became severely hypomethylated. However, the methylation-sensitive restriction enzyme HpaII revealed very low levels of modification at most of its recognition sites within the globin gene locus prior to treatment, and no further demethylation following treatment. Furthermore, in studies with hydroxyurea (HU) and cytosine-arabinoside, two other S-phase specific cytostatic/cytotoxic drugs, we have also observed induction of globin gene synthesis in K562 cells, and, with HU, expression of Beta-globin mRNA. The results suggest that these three drugs may act, at least in part, as chemical modifiers which affect the regulatory environment within cells, including trans-acting factors.